Abstract
Rapid diagnostics of bacterial infection is the key to successful recovery and eradication of the disease. Currently, identification of bacteria is based on the detection of highly abundant proteins, mainly ribosomal proteins, by routine MALDI-TOF mass spectrometry. However, relying solely on proteins is limited in subspecies typing for some pathogens. This is the case for, for example, the mycobacteria belonging to the Mycobacterium abscessus (MABS) complex, which is classified into three subspecies, namely, M. abscessus subsp. abscessus, M. abscessus subsp. bolletii, and M. abscessus subsp. massiliense. Being able to detect bacteria accurately and rapidly at the subspecies level could not only reliably identify the pathogen causing the disease but also enable better antibiotic stewardship. For instance, M. abscessus subsp. abscessus and M. abscessus subsp. bolletii possess a functional erm41 (erythromycin ribosomal methylation gene 41) gene, whilst M. abscessus subsp. massiliense does not, resulting in differences in macrolide antibiotic (e.g., clarithromycin and azithromycin) susceptibilities. This presents a challenge for physicians when designing an appropriate treatment regimen. To address this challenge, in addition to proteins, species-specific lipids have now been considered as a game changer in clinical microbiology diagnostics. However, their extraction can be time-consuming, and analysis requires the use of apolar toxic organic solvents (e.g., chloroform). Here, we present a new method to accurately detect species and subspecies, allowing the discrimination of the mycobacteria within the MABS complex and relying on the use of ethanol. We found that a combination of the matrix named super-DHB with 25% ethanol with a bacterial suspension at McFarland 20 gave robust and reproducible data, allowing the discrimination of the bacteria within the MABS complex strains tested in this study (n = 9). Further investigations have to be conducted to validate the method on a larger panel of strains for its use in diagnostic laboratories.
Highlights
Matrix-assisted laser desorption ionization/time-of-flight (MALDI-TOF) mass spectrometry (MS), a cost-effective, rapid, and accurate method for microbial identification based on protein signatures, has revolutionized microbiological diagnostics (Croxatto et al, 2012; Clark et al, 2013; Singhal et al, 2015)
We investigated the use of ethanol (CH3CH2OH, relative polarity 0.654) as a commonly used organic solvent widely used in routine clinical microbiology laboratories worldwide as an alternative to chloroform and methanol to extract species-specific lipids for the rapid identification of bacteria belonging to the Mycobacterium abscessus (MABS) complex
We optimized the number of bacteria loaded onto the MALDI target plate and the solvent to solubilize the matrix prior to MS analyses in the positive ion mode to address the major challenge in subspecies typing by MALDI-TOF MS inside the MABS complex
Summary
Matrix-assisted laser desorption ionization/time-of-flight (MALDI-TOF) mass spectrometry (MS), a cost-effective, rapid, and accurate method for microbial identification based on protein signatures, has revolutionized microbiological diagnostics (Croxatto et al, 2012; Clark et al, 2013; Singhal et al, 2015). Massiliense does not (Kim et al, 2010; Ryu et al, 2016; Ryan and Byrd, 2018). This results in different macrolide antibiotic (i.e., clarithromycin and azithromycin) susceptibilities. Bolletii isolates (Adekambi et al, 2006; Esther et al, 2010; Kim et al, 2010; Koh et al, 2011; Harada et al, 2012) This presents a challenge for physicians when designing an appropriate treatment regimen (Koh et al, 2011; Brown-Elliott et al, 2012; Harada et al, 2012; Kothavade et al, 2013)
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